MPRAsnakeflow experiment QC report

Good Spearman correlation across replicates!

The data has a good Spearman correlations across replicates using a barcode threshold per oligo of 10! Spearman correlation of DNA is 1.00, RNA is 0.98 and DNA/RNA ratio is 0.96.

DNA over RNA counts

Plotting normalized counts of DNA vs RNA (median across replicates). Only oligos within all replicates are shown. We should see a variation within the RNA count data (along the y axis). If count data between RNA and DNA is highly correlated (e.g. follows the identity line) there is no variation between designed oligos. This is an indication that RNA is inflated with DNA and the DNA digestion before creating cDNA did not work as expected.

Oligo correlation

Oligo correlation plots of DNA, RNA and DNA/RNA ratios across replicates. First tab shows plots using (in average) 2302 oligos with a minimum number of 10 barcodes. Second tab shows all 2391 oligos that have assigned barcodes.

Condition	A	B	#Oligos A	#Oligos B	#Oligos Joined	DNA spearman	RNA spearman	Ratio spearman	DNA log2 pearson	RNA log2 pearson	Ratio log2 pearson
HEPG2	1	2	2303	2302	2302	1.00	0.98	0.96	1.00	0.98	0.96
HEPG2	1	3	2303	2303	2303	1.00	0.98	0.97	1.00	0.98	0.97
HEPG2	2	3	2302	2303	2302	1.00	1.00	0.99	1.00	1.00	0.99

Condition	A	B	#Oligos A	#Oligos B	#Oligos Joined	DNA spearman	RNA spearman	Ratio spearman	DNA log2 pearson	RNA log2 pearson	Ratio log2 pearson
HEPG2	1	2	2391	2391	2391	0.99	0.98	0.96	0.99	0.98	0.95
HEPG2	1	3	2391	2391	2391	0.99	0.98	0.97	0.99	0.98	0.97
HEPG2	2	3	2391	2391	2391	0.99	0.99	0.99	0.99	0.99	0.98

Experiment statistic

The total number of oligos in this experiment is 2398 (defined by the assignment) with 938503 associated barcodes.

In average across replicates we see 2391 from 458815 average barcodes in the count data and around 377427 barcodes where not in the assignment.

condition	replicate	oligos dna/rna	matched barcodes	unknown barcodes dna/rna	% matched barcodes	total dna counts	total rna counts	avg dna counts per bc	avg rna counts per bc	avg dna/rna barcodes per oligo
HEPG2	1	2391	458254	376620	54.89	21004761	55171411	25.16	66.08	191.66
HEPG2	2	2391	455257	295377	60.65	20013665	36062289	26.66	48.04	190.40
HEPG2	3	2391	462933	460283	50.14	29614890	55689543	32.08	60.32	193.61

Experiment	Barcodes	Counts	Average counts	Assigned barcodes	Assigned counts	Average assigned counts	Fraction assigned barcodes	Fraction assigned counts
HEPG2_1_DNA	1712608	21963588	12.82	469619	17029932	36.26	0.27	0.78
HEPG2_2_DNA	1643652	20988652	12.77	467754	16298931	34.85	0.28	0.78
HEPG2_3_DNA	2111501	30931153	14.65	477876	24015191	50.25	0.23	0.78
HEPG2_1_RNA	2778818	57511408	20.70	487102	43587667	89.48	0.18	0.76
HEPG2_2_RNA	2198428	37761946	17.18	482090	28544055	59.21	0.22	0.76
HEPG2_3_RNA	2777175	57875595	20.84	487794	43863941	89.92	0.18	0.76

Number of barcodes per oligo

Histogramm of number of barcodes per oligo. Median is blue, mean is red.

Activity

Violin and box plots of the log2 fold change of all oligos by the assay. Grouped by labels if set, otherwise NA. First tab shows plots using (in average) 2302 oligos with a minimum number of 10 barcodes. Second tab shows all 2391 oligos that have assigned barcodes.

--- title: "MPRAsnakeflow experiment QC report" page-layout: full format: html: code-fold: true code-tools: true embed-resources: true anchor-sections: true smooth-scroll: true toc: true theme: light: flatly dark: darkly jupyter: python3 --- ```{python} #| echo: false # Imports from IPython.display import display, Image, Markdown import pandas as pd ``` ```{python} #| tags: [parameters] #| echo: false ``` ```{python} #| echo: false oligo_cor_merged_table_thresh = pd.read_csv(f"{workdir}/{statistics_all_oligo_cor_thresh}", sep='\t') oligo_cor_merged_table_all = pd.read_csv(f"{workdir}/{statistics_all_oligo_cor_all}", sep='\t') dna_spear = oligo_cor_merged_table_thresh['DNA_spearman'].min() rna_spear = oligo_cor_merged_table_thresh['RNA_spearman'].min() ratio_spear = oligo_cor_merged_table_thresh['Ratio_spearman'].min() ``` ```{python} #| echo: false threshold_dna_spear = 0.85 threshold_rna_spear = 0.95 threshold_ratio_spear = 0.75 important = dna_spear < threshold_dna_spear and rna_spear < threshold_rna_spear and ratio_spear < threshold_ratio_spear warning = dna_spear < threshold_dna_spear or rna_spear < threshold_rna_spear or ratio_spear < threshold_ratio_spear if important: display(Markdown(f''' ::: {"{.callout-important}"} ## Low correlation! Spearman correlation across replicates is low using a barcode threshold per oligo of {thresh}! Spearman correlation of DNA is {dna_spear:.2f}, RNA is {rna_spear:.2f} and DNA/RNA ratio is {ratio_spear:.2f}. Minimum allowed values are {threshold_dna_spear:.2f}, {threshold_rna_spear:.2f}, and {threshold_ratio_spear:.2f}. ::: ''')) elif warning: display(Markdown(f''' ::: {"{.callout-warning}"} ## Low correlation warning! At least one (not all) Spearman correlations across replicates is low using a barcode threshold per oligo of {thresh}! Spearman correlation of DNA is {dna_spear:.2f}, RNA is {rna_spear:.2f} and DNA/RNA ratio is {ratio_spear:.2f}. Minimum allowed values are {threshold_dna_spear:.2f}, {threshold_rna_spear:.2f}, and {threshold_ratio_spear:.2f}. ::: ''')) else: display(Markdown(f''' ::: {"{.callout-tip}"} ## Good Spearman correlation across replicates! The data has a good Spearman correlations across replicates using a barcode threshold per oligo of {thresh}! Spearman correlation of DNA is {dna_spear:.2f}, RNA is {rna_spear:.2f} and DNA/RNA ratio is {ratio_spear:.2f}. ::: ''')) ``` # DNA over RNA counts Plotting normalized counts of DNA vs RNA (median across replicates). Only oligos within all replicates are shown. We should see a variation within the RNA count data (along the y axis). If count data between RNA and DNA is highly correlated (e.g. follows the identity line) there is no variation between designed oligos. This is an indication that RNA is inflated with DNA and the DNA digestion before creating cDNA did not work as expected. ::: {.panel-tabset} ## Threshold `{python} thresh` ```{python} #| echo: false Image(f"{workdir}/{dna_over_rna_thresh_plot}") ``` ## All ```{python} #| echo: false Image(f"{workdir}/{dna_over_rna_plot}") ``` ::: # Oligo correlation Oligo correlation plots of DNA, RNA and DNA/RNA ratios across replicates. First tab shows plots using (in average) `{python} "%d" % oligo_cor_merged_table_thresh['number_Oligos_Joined'].mean()` oligos with a minimum number of `{python} thresh` barcodes. Second tab shows all `{python} "%d" % oligo_cor_merged_table_all['number_Oligos_Joined'].mean()` oligos that have assigned barcodes. ```{python} #| echo: false def filter_table(df): df = df.round(2).drop(columns=['DNA_pearson','RNA_pearson', 'Ratio_pearson', 'fraction_Oligos_ReplicateA', 'fraction_Oligos_ReplicateB', 'NormSymmetry']) df.columns = df.columns.str.replace("_", " ", regex=False).str.replace("Replicate", " ", regex=False).str.replace("number ", "#", regex=False) return(df) ``` ::: {.panel-tabset} ## Threshold `{python} thresh` ```{python} #| echo: false oligo_cor_merged_table_thresh_filter = filter_table(oligo_cor_merged_table_thresh) oligo_cor_merged_table_thresh_filter.style.hide(axis="index").format( precision=2) ``` ## All ```{python} #| echo: false oligo_cor_merged_table_all_filter = filter_table(oligo_cor_merged_table_all) oligo_cor_merged_table_all_filter.style.hide(axis="index").format( precision=2) ``` ::: ::: {.panel-tabset} ## Threshold `{python} thresh` ::: {.panel-tabset} ### DNA ```{python} #| echo: false display(Image(f"{workdir}/{dna_oligo_coor_min_thre_plot }")) ``` ### RNA ```{python} #| echo: false display(Image(f"{workdir}/{rna_oligo_coor_min_thre_plot}")) ``` ### Ratio ```{python} #| echo: false display(Image(f"{workdir}/{ratio_oligo_min_thre_plot}")) ``` ::: ## All ::: {.panel-tabset} ### DNA ```{python} #| echo: false display(Image(f"{workdir}/{dna_oligo_coor_plot}")) ``` ### RNA ```{python} #| echo: false display(Image(f"{workdir}/{rna_oligo_coor_plot}")) ``` ### Ratio ```{python} #| echo: false display(Image(f"{workdir}/{ratio_oligo_coor_plot}")) ``` ::: ::: # Experiment statistic ```{python} #| echo: false df_merged = pd.read_csv(f"{workdir}/{statistics_all_merged}", sep='\t').round(2) n_oligos = df_merged['oligos design'].iloc[0] n_barcodes = df_merged['barcodes design'].iloc[0] avg_oligos = df_merged['oligos dna/rna'].mean() avg_matched_barcodes = df_merged['matched barcodes'].mean() avg_unknown_barcodes = df_merged['unknown barcodes dna/rna'].mean() ``` The total number of oligos in this experiment is `{python} f"{n_oligos}"` (defined by the assignment) with `{python} f"{n_barcodes}"` associated barcodes. In average across replicates we see `{python} f"{avg_oligos:.0f}"` from `{python} f"{avg_matched_barcodes:.0f}"` average barcodes in the count data and around `{python} f"{avg_unknown_barcodes:.0f}"` barcodes where not in the assignment. ::: {.panel-tabset} # DNA/RNA merged ```{python} #| echo: false df_merged.drop(columns=['oligos design','barcodes design', 'barcodes dna/rna'], inplace=True) df_merged.style.hide(axis="index").format( precision=2) ``` # DNA/RNA individual ```{python} #| echo: false df_single = pd.read_csv(f"{workdir}/{statistics_all_single}", sep='\t').round(2) df_single.style.hide(axis="index").format( precision=2) ``` ::: # Number of barcodes per oligo Histogramm of number of barcodes per oligo. Median is blue, mean is red. ::: {.panel-tabset} # DNA ```{python} #| echo: false display(Image(f"{workdir}/{counts_per_oligo_dna}")) ``` # RNA ```{python} #| echo: false display(Image(f"{workdir}/{counts_per_oligo_rna}")) ``` ::: # Activity Violin and box plots of the log2 fold change of all oligos by the assay. Grouped by labels if set, otherwise `NA`. First tab shows plots using (in average) `{python} "%d" % oligo_cor_merged_table_thresh['number_Oligos_Joined'].mean()` oligos with a minimum number of `{python} thresh` barcodes. Second tab shows all `{python} "%d" % oligo_cor_merged_table_all['number_Oligos_Joined'].mean()` oligos that have assigned barcodes. ::: {.panel-tabset} ## Threshold `{python} thresh` ```{python} #| echo: false display(Image(f"{workdir}/{activity_thresh}")) ``` ## All ```{python} #| echo: false display(Image(f"{workdir}/{activity_all}")) ``` :::